Resistance to thyroid hormone (RTH) is caused mainly by mutations of the thyroid hormone receptor (TR) β gene. Although, in vitro, TRα1 and TRβ1 mutants exhibit similar dominant negative effects against wild-type TR, no TRα mutants have ever been identified in RTH patients. It has been postulated that mutations in TRα gene may be lethal, compensated completely by intact TRβ or associated with phenotypic manifestations different from RTH. To investigate the consequences of mutant TRα1 expression in vivo, we tried to generate two different lines of transgenic mice which express a strong or a weak dominant negative mutant TR α1, respectively. First, we expressed βF451X identified in a patient with severe RTH and αF397X, which has an identical C-terminal truncation and a similarly strong dominant negative potency to βF451X, under the control of human polypeptide chain elongation factor 1α promoter. Six βF451X-transgenic mice were born from 223 transferred embryos, giving a transgenic frequency of 2.7%. By contrast, expression of αF397X resulted in quite a low transgenic frequency with 0.39% of the transferred embryos bearing the transgene. Only three transgenic mice were born with no apparently overt abnormalities, of which one male produced F1 offspring. The transgenic progeny expressed αF397X in the testis but we did not succeed in generating transgenic mice expressing αF397X in multiple organs. To avoid toxic effects mediated by a strong dominant negative activity of mutant TRα1, we exchanged αF397X for αK389E, which has an identical missense mutation and a relatively weak transdominant potency as βK443E identified in a patient with mild RTH. When expressed by cytomegalovirus immediate early enhancer-chicken β-actin promoter, we did not succeed in creating αK389E-transgenic mice despite three independent transgene-injections. These findings define crucial in vivo functions of mutant TRα1s during mouse fetal development and suggest the possibility that the expression of a dominant negative mutant TRα1 in extensive tissues from the early embryonal stages might be lethal.